Raccoonpox virus (RPV) belongs to the orthopoxvirus family, like vaccinia virus, but has an advantage in that it is naturally attenuated and appears to be native to North America. Unlike swinepox virus whose infection in non-porcine species is non-productive, raccoonpox virus infection is productive, but may not spread systemically like vaccinia virus.
Raccoonpox virus was originally isolated from the upper respiratory tract of 2 of 92 apparently healthy raccoons in 1962 in Maryland (1). Significant HI (hemagglutinating-inhibiting) antibody titers for RPV were demonstrated in 23% [22/92] of raccoon sera indicating that RPV is indigenous in North America (2). Serological surveys testing sera from 593 raccoons in Delaware demonstrated that 4% were positive for RPV neutralizing antibodies, 12% were positive by ELISA and 2.5% of the sera were positive by both tests (10). RPV is classified as a orthopoxvirus based upon biological, serological and biophysical methods. RPV and volepox are the only orthopoxviruses thought to be native to North America. The Hind III DNA restriction map of RPV is the most divergent from other orthopoxvirus DNA maps, which are highly conserved, indicating that RPV has diverged from the other group members (4,6). Of 29 HindIII fragments from the RPV genome, none comigrated with HindIII fragments that were common to other examined orthopoxvirus DNA. RPV is naturally attenuated (10-100.times.) and avirulent compared with wild type vaccinia virus. There are four published RPV DNA sequences which include a Sal I end fragment (2.2 kb) (5), a hemagglutinin gene (1.5 kb) (7), a protein phosphatase sequence (0.5 kb; H1L ) (15), and the TK gene (834 bp) (16). RPV TK shows 87% amino acid homology to TK of vaccinia and 84.3% nucleotide homology. RPV HA shows 53% amino acid homology with vaccinia HA and 66% nucleotide homology. No other information regarding the genomic organization of RPV is known.
To date, recombinant raccoon poxviruses have been generated by insertion of foreign genes such as rabies virus glycoprotein into the TK locus of raccoon poxvirus (7-14). RPV recombinants expressing foreign viral antigens have been shown to elicit host protective immune responses in non-raccoon species including dogs, cats, and sheep with no negative effects observed.
The present invention provides a recombinant raccoon poxvirus comprising a foreign DNA inserted into the HindIII M region, HindIII N region or HindIII U region of the raccoonpox virus genome. The recombinant raccoon poxvirus is useful as a vaccine in mammalian and avian species. Prior to the present invention, it was unknown whether non-essential regions existed in the HindIII M, HindIII N, or HindIII U regions, and whether a stable recombinant raccoonpox virus could be isolated with foreign DNA insertions into these regions.